Nanoparticles with both electrocatalytic properties and photoluminescent properties, preparation methods thereof, and applications in miRNA detection and cell imaging

Abstract

The present invention discloses a nanoparticle having both electrocatalytic performance and photoluminescent properties, a preparation method thereof, and application in miRNA detection and cell imaging. The preparation method comprises the following steps: S1, synthesizing MXene quantum dots MQDs by a hydrothermal method: S2, synthesizing PBA@MQDs nanoparticles. The present invention provides a PBA@MQDs nanoparticle having both electrocatalytic performance and photoluminescent properties, and constructs a capture probe using a nucleic acid aptamer as a targeting unit, which can be used for high-sensitivity detection and intracellular imaging of miRNA; the nanoparticle has good electrocatalytic activity, controllable nanostructure and low cytotoxicity, and can realize accurate recognition and detection of miRNA through simple aptamer modification, and the detection range can reach 1 fM~1 nM; at the same time, the nanoparticle has the ability to enter cells to realize intracellular miRNA imaging analysis.

Description

Technical Field

[0001] The present invention relates to a field, and in particular to a nanoparticle having both electrocatalytic performance and photoluminescent properties, a preparation method thereof, and application in miRNA detection and cell imaging. Background Art

[0002] MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs with a length of about 22 nucleotides. They participate in and affect the growth, cell differentiation, proliferation and apoptosis of organisms by regulating gene expression, and can reflect the development and metastasis of tumors. They are an ideal tumor marker. MiRNAs can stably exist in biological serum or plasma, and have the advantages of high temperature resistance, acid and alkali resistance and long-term storage. Efficient and accurate detection of miRNAs and in situ analysis in tumor cells are the current research goals in clinical diagnosis. However, due to the low abundance of intracellular miRNAs, highly homologous sequences, and heterologous expression at different times or intracellular locations, accurate detection and in situ analysis of miRNAs remains a major challenge.

[0003] At present, the methods used for intracellular miRNAs analysis and imaging mainly include catalytic hairpin assembly hybridization chain reaction, nano-flash enzyme amplification, and enzyme-free DNA ring. These methods are more or less limited by the low sensitivity caused by the target equivalent reaction ratio, the amount of exogenous enzymes used, and the slow reaction kinetics. The two-dimensional material MXene has been widely studied in the field of biosensing due to its metal-like electrocatalytic properties. Converting it into quantum dots with quantum confinement and edge effects can give it luminescent properties, which can be further applied to the imaging of biological systems. Prussian blue (PBA) is a type of coordination compound formed by cyano groups and transition metal ions, which has the advantages of adjustable structure, high specific surface area and large porosity. Among them, PBA with dual metal ions as the center component exhibits different metal ion oxidation states and balanced charge vacancies compared to single metal centers, and has strong affinity and biocompatibility for biological molecules, making it an ideal material for the development of biosensors. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a nanoparticle with both electrocatalytic performance and photoluminescent properties, a preparation method thereof, and its application in miRNA detection and cell imaging in view of the deficiencies in the above-mentioned prior art. In the present invention, in order to achieve quantitative detection and intracellular in situ analysis of miRNAs, a single layer of Ti2CT is firstly prepared by a one-step hydrothermal method. x MXene nanosheets converted to Ti2CT xMXene quantum dots (MQDs) give it a photoluminescent effect. Subsequently, MQDs were electrostatically adsorbed and assembled on the surface of PBA nanoparticles coordinated by Co-Mn bimetallic ions using low-speed synthesis to synthesize PBA@MQDs nanoparticles with uniform particle size and uniform distribution. By modifying the recognition sequence of the target miRNA on the surface of PBA@MQDs nanoparticles, high-sensitivity detection of miRNA and identification of base mismatches were achieved, while imaging and analyzing miRNA in living cells.

[0005] To achieve the above object, the technical solution adopted by the present invention is: In a first aspect of the present invention, a method for preparing nanoparticles having both electrocatalytic properties and photoluminescent properties is provided, comprising the following steps:

[0006] S1. Synthesis of MXene quantum dots MQDs:

[0007] S1-1. Etching Ti2AlC MAX precursor with lithium fluoride hydrochloric acid solution to prepare Ti2CT x MXene powder;

[0008] S1-2, Ti2CT x MXene powder was dispersed in a solvent, stirred, centrifuged and cleaned to prepare a single layer of Ti2CT x The MXene suspension is then transferred to a reactor, ammonia water is added under stirring, and a hydrothermal reaction is carried out under heating to prepare MXene quantum dots: MQDs;

[0009] S2. Synthesis of PBA@MQDs nanoparticles:

[0010] The MQDs prepared in step S1 are prepared into a dispersion, and then a mixed solution of MnSO4 and K3[Co(CN)6] is added dropwise. After the addition is completed, the product is washed and dried to obtain PBA@MQDs nanoparticles, i.e., the nanoparticles having both electrocatalytic properties and photoluminescent properties.

[0011] Preferably, step S1 specifically comprises:

[0012] S1-1. Weigh the LiF reagent and dissolve it in the HCl solution. Stir at room temperature to obtain a hydrochloric acid solution of lithium fluoride. Add Ti2AlC MAX powder under stirring and react for 18-72 h. After the reaction, the product is centrifuged and washed with ethanol and deionized water in sequence until the pH value of the solution obtained by centrifugation reaches 5-7. Then, the solid product is vacuum dried to obtain Ti2CT x MXene powder;

[0013] S1-2, Ti2CT xMXene powder was added to dimethyl sulfoxide solvent, stirred at room temperature for 12-48 h, and then centrifuged and washed to obtain a single layer of Ti2CT x MXene suspension; ultrasonically disperse under N2 protection, then move into a reactor, add ammonia water while stirring at room temperature, stir evenly, and then hydrothermally react at 110-130°C for 3-12 h. After the reaction is completed, cool to room temperature, filter the product with a filter membrane, and rotary evaporate to obtain MXene quantum dots: MQDs.

[0014] Preferably, step S1 specifically comprises:

[0015] S1-1. Weigh 0.666 g of LiF reagent and dissolve it in 10 mL of 6 M HCl solution. Stir it at room temperature for 30 min. Place it in a constant temperature water bath at 35 °C. Add 1 g of Ti2AlC MAX powder while stirring. React for 36 h. The product is centrifuged and washed with ethanol and deionized water in sequence until the pH value of the solution obtained by centrifugation reaches 6. Then, the solid product is vacuum dried at 50 °C overnight to obtain Ti2CT x MXene powder;

[0016] S1-2, Ti2CT x MXene powder was added to dimethyl sulfoxide solvent to prepare a mixture with a concentration of 5 mg / mL. After stirring at room temperature for 24 h, it was centrifuged and washed to obtain a single layer of Ti2CT x MXene suspension; after ultrasonic treatment for 30 min under N2 protection, move it into the reactor, add 150 μL of 25% ammonia water under stirring at room temperature, stir evenly and then hydrothermally react at 120 °C for 6 h. After the reaction, cool to room temperature, filter the product with a 0.22 μm filter membrane and rotary evaporate to obtain MXene quantum dots: MQDs.

[0017] Preferably, step S2 specifically comprises:

[0018] S2-1, using a mixed solution of potassium chloride and HCl as a solvent, respectively preparing a MnSO4 solution and a K3[Co(CN)6] solution, and mixing the MnSO4 solution and the K3[Co(CN)6] solution to obtain a mixed solution of MnSO4 and K3[Co(CN)6];

[0019] S2-2. The MQDs prepared in step S1 are prepared into an aqueous solution, and then polyvinyl pyrrolidone is added and stirred to prepare a MQD mixed solution containing polyvinyl pyrrolidone, to which a mixed solution of MnSO4 and K3[Co(CN)6] is added dropwise. After the addition is completed, the product is centrifugally washed with deionized water and ethanol in turn, and vacuum dried to obtain PBA@MQDs nanoparticles, i.e., the nanoparticles having both electrocatalytic properties and photoluminescent properties.

[0020] Preferably, step S2 specifically comprises:

[0021] S2-1, using a mixed solution of 0.1 M potassium chloride (KCl) and 0.1 M HCl as a solvent, prepare 50 mL of a 0.005 M MnSO4 solution and 50 mL of a 0.01 M K3[Co(CN)6] solution, respectively, and mix equal volumes of the MnSO4 solution and the K3[Co(CN)6] solution to obtain a mixed solution of MnSO4 and K3[Co(CN)6];

[0022] S2-2. Take the MQDs prepared in step S1 to prepare an aqueous solution of MQDs with a concentration of 5 mg / mL. Take 10 mL of the aqueous solution of MQDs, add 0.01 M polyvinyl pyrrolidone thereto, stir for 5-10 min, and prepare a MQD mixed solution containing polyvinyl pyrrolidone. Add 25 mL of a mixed solution of MnSO4 and K3[Co(CN)6] dropwise thereto at a rate of 300 μL / min. After the addition is completed, the product is centrifugally washed with deionized water and ethanol in turn, and dried overnight in vacuum at 60°C to obtain PBA@MQDs nanoparticles, i.e., the nanoparticles having both electrocatalytic properties and photoluminescent properties.

[0023] The second aspect of the present invention provides a nanoparticle having both electrocatalytic performance and photoluminescent properties, which is prepared by the method as described above.

[0024] The third aspect of the present invention provides an application of the nanoparticles having both electrocatalytic properties and photoluminescent properties as described above in miRNA detection, wherein the application method is:

[0025] The electrode is modified with the nanoparticles having both electrocatalytic properties and photoluminescent properties as described above, and then the capture probe probe designed for the target miRNA is modified to obtain the probe-NH2-PBA@MQDs modified electrode, which is then used to detect the target miRNA using an electrochemical method.

[0026] Preferably, the method for applying the nanoparticles having both electrocatalytic properties and photoluminescent properties in miRNA detection is:

[0027] 1) Preparation of NH2-PBA@MQDs modified electrode:

[0028] 1-1) adding polyetherimide and the nanoparticles having both electrocatalytic properties and photoluminescent properties as described above into deionized water, stirring and reacting at room temperature, filtering after the reaction, and vacuum drying to obtain NH2-PBA@MQDs;

[0029] 1-2) Add NH2-PBA@MQDs to deionized water and disperse by ultrasonic to obtain NH2-PBA@MQDs solution;

[0030] 1-3) The substrate electrode is pretreated, and then the absorbed NH2-PBA@MQDs solution is dropped onto the surface of the substrate electrode, and after drying, the NH2-PBA@MQDs modified electrode is obtained;

[0031] 2) Preparation of probe-NH2-PBA@MQDs modified electrode

[0032] 2-1) Design the corresponding capture probe according to the target miRNA, prepare a probe solution, and then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide for activation;

[0033] 2-2) Adding a fixing solution to the probe solution obtained in step 2-1) to obtain a probe modification solution, immersing the NH2-PBA@MQDs modified electrode in the probe modification solution for reaction, cleaning the electrode surface after the reaction, and then dropping a melanin-enriching hormone MCH blocking polypeptide solution on the electrode surface for co-incubation to obtain a probe-NH2-PBA@MQDs modified electrode;

[0034] 3) Target miRNA detection:

[0035] The sample solution to be tested was co-incubated with the probe-NH2-PBA@MQDs modified electrode to obtain a detection electrode, which was used as a working electrode. An electrochemical workstation was used for electrochemical analysis, and the concentration of the target miRNA in the sample solution was obtained based on the obtained current signal and a pre-constructed standard curve characterizing the relationship between the current signal and the concentration of the target miRNA.

[0036] Preferably, the method for applying the nanoparticles having both electrocatalytic properties and photoluminescent properties in miRNA detection is:

[0037] 1) Preparation of NH2-PBA@MQDs modified electrode:

[0038] 1-1) polyetherimide and the nanoparticles having both electrocatalytic properties and photoluminescent properties as described above are added to deionized water to prepare a mixed solution with a polyetherimide concentration of 5 mg / mL and a nanoparticle concentration of 1 mg / mL, stirred at room temperature for 4 h, filtered, and vacuum dried at 50°C for 12 h to obtain NH2-PBA@MQDs;

[0039] 1-2) Add NH2-PBA@MQDs to deionized water and disperse by ultrasonic to prepare a NH2-PBA@MQDs solution with a concentration of 1 mg / mL;

[0040] 1-3) The Au electrode was used as the base electrode, polished until the surface was smooth, rinsed with deionized water, and dried in N2 atmosphere. Then 10 μL of NH2-PBA@MQDs solution was dripped on the surface of the Au electrode. After natural drying, the solution was repeatedly dripped once to obtain the NH2-PBA@MQDs modified electrode.

[0041] 2) Preparation of probe-NH2-PBA@MQDs modified electrode

[0042] 2-1) Design the corresponding capture probe according to the target miRNA, prepare a probe solution with a concentration of 5 μM using MES buffer, then add 0.3 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.6 M N-hydroxysuccinimide (NHS), and activate at 4°C for 2 h;

[0043] 2-2) Add 5 μL of a fixing solution (10 mM Tris-HCl, 1 mM EDTA and 0.1 M NaCl, pH=7.4) to 5 μL of a 5 μM probe solution to obtain a probe modification solution, immerse the NH2-PBA@MQDs modified electrode in the probe modification solution and react at 4°C for 12 h, then wash the electrode surface with a 10 mM, pH=7.4 Tris-HCl solution, drop 5 μL of a 10 mM melanin-enriching hormone MCH blocked polypeptide solution on the electrode surface, and incubate at 4°C for 1 h to obtain a probe-NH2-PBA@MQDs modified electrode;

[0044] 3) Target miRNA detection:

[0045] The sample solution to be tested was incubated with the probe-NH2-PBA@MQDs modified electrode at 37°C to obtain a detection electrode. The detection electrode was used as a working electrode. Electrochemical analysis was performed using an electrochemical workstation. The concentration of the target miRNA in the sample solution was obtained based on the obtained current signal and a pre-constructed standard curve characterizing the relationship between the current signal and the concentration of the target miRNA.

[0046] In a fourth aspect of the present invention, there is provided an application of the nanoparticles having both electrocatalytic properties and photoluminescent properties as described above in cell imaging, wherein the application method is:

[0047] The capture probe probe designed for the target miRNA is modified on the nanoparticles having both electrocatalytic properties and photoluminescent properties as described above to obtain the aptamer-modified nanoparticles probe-NH2-PBA@MQDs, which are then used as dyes for fluorescence imaging of the target miRNA in cells.

[0048] Preferably, the aptamer-modified nanoparticle probe-NH2-PBA@MQDs is prepared by the following method:

[0049] The corresponding capture probe was designed according to the target miRNA, and a probe solution with a concentration of 5 μM was prepared using MES buffer. Then, 0.3 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.6 M N-hydroxysuccinimide (NHS) were added and activated at 4°C for 2 h.

[0050] 5 μL of the fixing solution was added to 5 μL of the 5 μM probe solution to obtain the probe modification solution. 1 mg / mL of the nanoparticles having both electrocatalytic properties and photoluminescent properties as described above were added to the obtained probe modification solution and reacted for 12 h. The obtained nanoparticles were then washed with 10 mM Tris-HCl solution and incubated with 5 μL of 10 mM MCH solution for 1 h to obtain the aptamer-modified nanoparticles probe-NH2-PBA@MQDs.

[0051] Preferably, the target miRNA is miRNA-141, and its sequence is: 5′-UAACACUGUCUGGUAAAGAUGG-3′;

[0052] The sequence of the capture probe probe for the target miRNA is: 5′-COOH-CCATCTTTACCAGACAGTGTTA-3′.

[0053] The beneficial effects of the present invention are:

[0054] The present invention provides a PBA@MQDs nanoparticle having both electrocatalytic performance and photoluminescent properties, and constructs a capture probe with a nucleic acid aptamer as a targeting unit, which can be used for high-sensitivity detection and intracellular imaging of miRNA; the PBA@MQDs nanoparticle has good electrocatalytic activity, controllable nanostructure and low cytotoxicity, and can achieve accurate recognition and detection of miRNA through simple aptamer modification, with a detection range of 1fM~1nM; at the same time, the nanoparticle has the ability to enter cells to realize intracellular miRNA imaging analysis, and is expected to provide a new solution for real-time and in situ analysis of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Schematic diagram of the preparation of PBA@MQDs nanoparticles in the present invention and their application in the detection and imaging of miRNAs;

[0056] Figure 2 Morphological characterization of PBA@MQDs nanoparticles prepared in Example 1: (a) SEM; (b) TEM; (c) lattice diffraction; (d) HRTEM;

[0057] Figure 3 Structural characterization of PBA@MQDs nanoparticles prepared in Example 1: (a) C1s spectrum of PBA@MQDs nanoparticles; (b) FTIR spectra of PBA, PBA@MQDs and NH2-PBA@MQDs.

[0058] Figure 4 Photoluminescence and cell safety evaluation of PBA@MQDs nanoparticles prepared in Example 1: (a) UV-vis absorption spectrum and fluorescence spectrum; (b) cytotoxicity.

[0059] Figure 5 Electrochemical detection performance test results of probe-NH2-PBA@MQDs modified electrode for miRNA-141: (a) SWV current response of miRNA-141 at different concentrations; (b) linear curve.

[0060] Figure 6 The probe / NH2-PBA@MQDs modified electrode was used to detect the current response signals of different miRNAs;

[0061] Figure 7Results of imaging experiments of intracellular miRNA using PBA@MQDs and probe-NH2-PBA@MQDs nanoparticles: Hoechst-labeled cell nucleus, (a) PBA@MQDs; (b) probe-NH2-PBA@MQDs+miRNA-141 inhibitor; (c) probe-NH2-PBA@MQDs. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0063] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0064] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0065] The nucleic acid sequences involved in the following examples are as follows:

[0066] Table 1

[0067] Example 1

[0068] A nanoparticle having both electrocatalytic performance and photoluminescent properties, wherein the preparation method comprises the following steps:

[0069] S1. Synthesis of MXene quantum dots MQDs:

[0070] S1-1, weigh 0.666 g LiF reagent and dissolve it in 10 mL 6 M HCl solution, stir at room temperature for 30 min, place in a constant temperature water bath at 35 ° C, add 1 g Ti2AlC MAX powder (purchased from Jilin Yiyi Technology Co., Ltd., 200 mesh) under stirring, react for 36 h, centrifuge and wash the product with ethanol and deionized water in sequence until the pH value of the solution obtained by centrifugation reaches 6, and then vacuum dry the solid product at 50 ° C overnight to obtain Ti2CT x MXene powder;

[0071] S1-2, Ti2CT xMXene powder was added to dimethyl sulfoxide solvent to prepare a mixture with a concentration of 5 mg / mL. After stirring at room temperature for 24 h, it was centrifuged and washed to obtain a single layer of Ti2CT x MXene suspension; after ultrasonic treatment for 30 minutes under N2 protection, move it into the reactor, add 150 μL of 25% ammonia water under stirring at room temperature, stir evenly and then hydrothermally react at 120 °C for 6 hours. After the reaction, cool to room temperature, filter the product with a 0.22 μm filter membrane and rotary evaporate to obtain MXene quantum dots: MQDs.

[0072] S2. Synthesis of PBA@MQDs nanoparticles:

[0073] S2-1, using a mixed solution of 0.1 M potassium chloride (KCl) and 0.1 M HCl as a solvent, prepare 50 mL of a 0.005 M MnSO4 solution and 50 mL of a 0.01 M K3[Co(CN)6] solution, respectively, and mix equal volumes of the MnSO4 solution and the K3[Co(CN)6] solution to obtain a mixed solution of MnSO4 and K3[Co(CN)6];

[0074] S2-2. Take the MQDs prepared in step S1 to prepare an aqueous solution of MQDs with a concentration of 5 mg / mL. Take 10 mL of the aqueous solution of MQDs, add 0.01 M polyvinyl pyrrolidone thereto, stir for 10 min, and prepare a MQD mixed solution containing polyvinyl pyrrolidone. Add 25 mL of a mixed solution of MnSO4 and K3[Co(CN)6] dropwise thereto at a rate of 300 μL / min. After the addition is completed, the product is centrifugally washed with deionized water and ethanol in turn, and dried overnight in vacuum at 60°C to obtain PBA@MQDs nanoparticles, i.e., nanoparticles having both electrocatalytic properties and photoluminescent properties.

[0075] Reference Figure 2 , morphology characterization of PBA@MQDs nanoparticles prepared in Example 1: (a) SEM; (b) TEM; (c) lattice diffraction; (d) HRTEM.

[0076] In this example, PBA@MQDs nanoparticles with an average particle size of less than 100 nm were synthesized and prepared. MQDs were wrapped on the surface of nanocubic PBA to form a core-shell structure. Lattice diffraction and HRTEM characterization showed that PBA had a single crystal structure, and the (011) face of PBA and the (101) face of MQDs appeared in the lattice of PBA@MQDs nanoparticles, indicating the successful synthesis of PBA@MQDs nanoparticles.

[0077] Reference Figure 3, structural characterization of PBA@MQDs nanoparticles prepared in Example 1: (a) C1s spectrum of PBA@MQDs nanoparticles; (b) FTIR spectra of PBA, PBA@MQDs and NH2-PBA@MQDs.

[0078] The elemental valence state of PBA@MQDs was analyzed by XPS narrow spectrum characterization of C1s. The C≡N bond of PBA appeared at 282.2 eV. The four peaks of 281.8 eV (C-Ti), 284.6 eV (CC), 286.7 eV (CO) and 288.1 eV (OC=O) further proved the formation of the MQDs adsorption layer on the PBA surface, indicating the successful preparation of PBA@MQDs. Figure 3 FTIR characterization of b at 3410 cm -1 The stretching vibration of -OH and NH vibration between water molecules indicate the successful modification of the surface amino of PBA@MQDs.

[0079] Reference Figure 4 , Photoluminescence and cell safety evaluation of PBA@MQDs nanoparticles prepared in Example 1: (a) UV-vis absorption spectrum and fluorescence spectrum; (b) cytotoxicity.

[0080] The cell safety evaluation method is:

[0081] A549 lung cancer cells were selected as the imaging cell model of miRNA-141 in the present invention for culture. The cells were cultured to the logarithmic growth phase. When the cells were in good condition, they were inoculated in a 96-well cell culture plate and incubated for 24 h. After washing with PBS, different concentrations of PBA@MQDs nanoparticles (0-2.0 mg / mL) were added and continued to be incubated for 24 h. After the incubation, they were washed with PBS, and then 100 μL of 10% MTT solution was added and incubated for 40 min. The absorbance value (OD) at 490 nm was read using a microplate reader to evaluate the cell safety.

[0082] The absorption and emission wavelengths of PBA@MQDs nanoparticles were characterized and analyzed by UV-vis absorption and fluorescence spectroscopy. Figure 4 As shown in a, PBA@MQDs nanoparticles have characteristic absorption peaks at 260 nm and 320 nm wavelengths, and the emission wavelength is 440 nm, proving that PBA@MQDs nanoparticles emit blue fluorescence and have fluorescence imaging properties. The cytotoxicity of PBA@MQDs nanoparticles was characterized by the MTT method. The results are shown in Figure 4 As shown in (b), the cell activity of A549 cells after co-incubation with different concentrations of PBA@MQDs nanoparticles was above 80%, indicating that the nanoparticles have good cell safety and can be applied to cell systems. Example 2

[0083] An application of PBA@MQDs nanoparticles in miRNA detection, the application method is:

[0084] 1) Preparation of NH2-PBA@MQDs modified electrode:

[0085] 1-1) polyetherimide and the PBA@MQDs nanoparticles prepared in Example 1 were added to deionized water to prepare a mixed solution with a polyetherimide concentration of 5 mg / mL and a nanoparticle concentration of 1 mg / mL, stirred at room temperature for 4 h, filtered, and vacuum dried at 50°C for 12 h to obtain NH2-PBA@MQDs;

[0086] 1-2) Add NH2-PBA@MQDs to deionized water and disperse by ultrasonic to prepare a NH2-PBA@MQDs solution with a concentration of 1 mg / mL;

[0087] 1-3) The Au electrode was used as the base electrode, polished until the surface was smooth, rinsed with deionized water, and dried in N2 atmosphere. Then 10 μL of NH2-PBA@MQDs solution was dripped on the surface of the Au electrode. After natural drying, the solution was repeatedly dripped once to obtain the NH2-PBA@MQDs modified electrode.

[0088] 2) Preparation of probe-NH2-PBA@MQDs modified electrode:

[0089] 2-1) Design the corresponding capture probe according to the target miRNA, use MES buffer (pH=6.0, purchased from Beijing Bioson Biotechnology Co., Ltd.) to prepare a probe solution with a concentration of 5 μM, then add 0.3 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.6 M N-hydroxysuccinimide (NHS), and activate at 4°C for 2 hours;

[0090] 2-2) Add 5 μL of fixing solution (10 mM Tris-HCl, 1 mM EDTA and 0.1 M NaCl, pH=7.4) to 5 μL of 5 μM probe solution to obtain probe modification solution, immerse the NH2-PBA@MQDs modified electrode in the probe modification solution and react at 4°C for 12 h, then use 10 mM Tris-HCl solution, pH=7.4 to clean the electrode surface, add 5 μL of 10 mM melanin enrichment hormone MCH blocking peptide solution (Beijing Bioson Biotechnology Co., Ltd., bs-18784P) on the electrode surface, incubate at 4°C for 1 h, and obtain probe-NH2-PBA@MQDs modified electrode;

[0091] 3) Target miRNA detection:

[0092] The sample solution to be tested was incubated with the probe-NH2-PBA@MQDs modified electrode at 37°C to obtain a detection electrode. The detection electrode was used as a working electrode. Electrochemical analysis was performed using an electrochemical workstation. The concentration of the target miRNA in the sample solution was obtained based on the obtained current signal and a pre-constructed standard curve characterizing the relationship between the current signal and the concentration of the target miRNA.

[0093] In this example, the target miRNA is miRNA-141, and its sequence is: 5′-UAACACUGUCUGGUAAAGAUGG-3′;

[0094] The sequence of the capture probe probe for the target miRNA is: 5′-COOH-CCATCTTTACCAGACAGTGTTA-3′.

[0095] In this example, miRNA-141 (5′-UAACACUGUCUGGUAAAGAUGG-3′) was used as the target detection object, and miRNA-141 solutions of different concentrations were prepared with H-buffer buffer (10 mM Tris-HCl, 1 mM EDTA and 0.2 M NaCl, pH = 7.4). The probe-NH2-PBA@MQDs modified electrode prepared above was incubated with the miRNA-141 solution at 37 °C for 6 h to obtain the miRNA-141@probe-NH2-PBA@MQDs modified electrode, which was stored for later use.

[0096] The sensor analysis test was carried out on a CHI 660E electrochemical workstation, with a saturated silver-silver chloride electrode (Ag / AgCl) as the reference electrode, a platinum electrode as the counter electrode, and miRNA-141@probe-NH2-PBA@MQDs as the working electrode. Square wave voltammetry (SWV) was used as the electrochemical test method, the electrolyte was a 10 mM K3[Fe(CN)6] solution containing 0.1 M KCl, the potential scanning range was -0.1~0.6 V, and the increment E was 0.004 V. The SWV response of the probe / NH2-PBA@MQDs modified electrode co-incubated with different concentrations of miRNA-141 was tested to obtain the corresponding current signal; the logarithm of the current concentration was used as the abscissa and the corresponding miRNA-141 concentration was used as the ordinate, and a linear fit was performed to obtain the standard curve.

[0097] In addition, miRNA-200a (5′-UAACACUGUCUGGUAACGAUGU-3′), miRNA-155 (5′-UUAAUGCUAAUCGUGAUAGGGGU-3′), miRNA-21 (5′-UAGCUUAUCAGACUGAUGUUGA-3′), and miRNA-182 (5′-UUUGGCAAUGGUAGAACUCACACU-3′) were selected as comparison sequences and subjected to the same incubation test to analyze the specific base adaptation performance of the electrode.

[0098] Reference Figure 5 , are the test results of the electrochemical detection performance of probe-NH2-PBA@MQDs modified electrode for miRNA-141: (a) SWV current response of different concentrations of miRNA-141; (b) linear curve.

[0099] The prepared probe-NH2-PBA@MQDs modified electrode was used for electrochemical test of different concentrations of miRNA-141 (1 fM~1nM), and the following results were obtained: Figure 5 The SWV current response curve shown in a was fitted with the peak current and the corresponding miRNA-141 concentration to obtain Figure 5 b shows the linear calibration curve. As can be seen from the figure, the peak current and the corresponding miRNA-141 concentration logarithm show a good linear relationship, and its linear equation is I=2.73+159.5logC in the range of miRNA-141 concentration of 1 fM~1 nM. miRNA (R 2 =0.994).

[0100] Reference Figure 6 , the probe / NH2-PBA@MQDs modified electrode is used to detect the current response signals of different miRNAs.

[0101] Under the same conditions of the above miRNA-141 detection parameters, the prepared probe-NH2-PBA@MQDs modified electrode was used for miRNAs with different nucleic acid sequences (including miRNA-182, miRNA-21, miRNA-155 and miRNA-200a) for comparative analysis. Figure 6 As shown, it can be seen that the interference of the current response of other miRNAs on the detection signal is negligible, indicating that the modified electrode has good mismatch discrimination ability. Example 3

[0102] An application of PBA@MQDs nanoparticles in cell imaging, the application method is:

[0103] The capture probe probe designed for the target miRNA is modified on the PBA@MQDs nanoparticles prepared in Example 1 to obtain nucleic acid aptamer-modified nanoparticles probe-NH2-PBA@MQDs, and then the nucleic acid aptamer-modified nanoparticles are used as a dye for fluorescent imaging of the target miRNA in cells.

[0104] Wherein, the nucleic acid aptamer-modified nanoparticle probe-NH2-PBA@MQDs is prepared by the following method:

[0105] The corresponding capture probe was designed according to the target miRNA, and a probe solution with a concentration of 5 μM was prepared using MES buffer. Then, 0.3 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.6 M N-hydroxysuccinimide (NHS) were added and activated at 4°C for 2 h.

[0106] 5 μL of the fixing solution was added to 5 μL of the 5 μM probe solution to obtain the probe modification solution, and 1 mg / mL of the nanoparticles having both electrocatalytic properties and photoluminescent properties prepared in Example 1 was added to the obtained probe modification solution, and the reaction was carried out for 12 h. Then, the obtained nanoparticles were washed with 10 mM Tris-HCl solution, and the nanoparticles were incubated with 5 μL of 10 mM MCH solution for 1 h to obtain the nucleic acid aptamer-modified nanoparticles probe-NH2-PBA@MQDs.

[0107] Among them, the target miRNA is miRNA-141, and its sequence is: 5′-UAACACUGUCUGGUAAAGAUGG-3′; the sequence of the capture probe probe for the target miRNA is: 5′-COOH-CCATCTTTACCAGACAGTGTTA-3′.

[0108] PBA@MQDs and probe-NH2-PBA@MQDs nanoparticles were used in cell imaging experiments of miRNAs:

[0109] Select the A549 cells (10 5cells / mL) were inoculated in a confocal culture dish and placed in a cell culture incubator for 24 h, and then the PBA@MQDs prepared in Example 1 and the probe-NH2-PBA@MQDs prepared above were added and incubated for 4 h; at the same time, a control group was set up: under the same conditions, miRNA-141 inhibitor (miRNA-141 inhibitor, which can reduce the content of intracellular miRNA-141) was added and the above operation was repeated. After the incubation, PBS was washed three times, 4% paraformaldehyde was fixed at room temperature for 10 min, and then washed with PBS, Hoechst was added for staining for 15 min, and PBS was washed three times, and the imaging performance of miRNAs was evaluated by laser confocal scanning microscopy.

[0110] Reference Figure 7 , are the experimental results of using PBA@MQDs and probe-NH2-PBA@MQDs nanoparticles for intracellular miRNA imaging: Hoechst labeled cell nucleus, (a) PBA@MQDs; (b) probe-NH2-PBA@MQDs + miRNA-141inhibitor; (c) probe-NH2-PBA@MQDs.

[0111] The cell imaging performance was investigated by setting up three control groups to co-incubate with the cells. Figure 7 As shown, the nanoparticles modified with nucleic acid aptamers (probe-NH2-PBA@MQDs) showed bright blue fluorescence signals in areas other than the cell nucleus ( Figure 7 c), No fluorescence signal was observed in the cytoplasm after co-incubation of unmodified nanoparticles with cells ( Figure 7 a), indicating that the modification of the aptamer helps the nanoparticles to be taken up by cells and thus achieve miRNA imaging. In addition, in order to prove that the blue fluorescence signal in the cell comes from the intracellular miRNA, the miRNA-141 inhibitor was added to reduce the intracellular miRNA-141 content to examine its effect; Figure 7 As shown in b, the area outside the cell nucleus where the inhibitor was added showed weak blue fluorescence, proving that the fluorescence signal generated by probe / NH2-PBA@MQDs originated from the miRNA in the cell.

[0112] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A method for preparing nanoparticles having both electrocatalytic properties and photoluminescent properties, characterized in that: The following steps are involved: S1. Synthesis of MXene quantum dots MQDs: S1-1. Etching Ti2AlC MAX precursor with lithium fluoride hydrochloric acid solution to prepare Ti2CT x MXene powder; S1-2, Ti2CT x MXene powder was dispersed in a solvent, stirred, centrifuged and cleaned to prepare a single layer of Ti2CT x The MXene suspension is then transferred to a reactor, ammonia water is added under stirring, and a hydrothermal reaction is carried out under heating to prepare MXene quantum dots: MQDs; S2. Synthesis of PBA@MQDs nanoparticles: The MQDs prepared in step S1 are prepared into a dispersion, and then a mixed solution of MnSO4 and K3[Co(CN)6] is added dropwise. After the addition is completed, the product is washed and dried to obtain PBA@MQDs nanoparticles, i.e., the nanoparticles having both electrocatalytic properties and photoluminescent properties.

2. The method for preparing nanoparticles having both electrocatalytic performance and photoluminescent properties according to claim 1, characterized in that: Step S1 is specifically as follows: S1-1. Weigh the LiF reagent and dissolve it in the HCl solution. Stir at room temperature to obtain a hydrochloric acid solution of lithium fluoride. Add Ti2AlC MAX powder under stirring and react for 18-72 h. After the reaction, the product is centrifuged and washed with ethanol and deionized water in sequence until the pH value of the solution obtained by centrifugation reaches 5-7. Then, the solid product is vacuum dried to obtain Ti2CT x MXene powder; S1-2, Ti2CT x MXene powder was added to dimethyl sulfoxide solvent, stirred at room temperature for 12-48 hours, and then centrifuged and washed to obtain a single layer of Ti2CT x MXene suspension; ultrasonically disperse under N2 protection, then move into a reactor, add ammonia water while stirring at room temperature, stir evenly, and then hydrothermally react at 110-130°C for 3-12 h. After the reaction is completed, cool to room temperature, filter the product with a filter membrane, and rotary evaporate to obtain MXene quantum dots: MQDs.

3. The method for preparing nanoparticles having both electrocatalytic performance and photoluminescent properties according to claim 2, characterized in that: Step S1 is specifically as follows: S1-1. Weigh 0.666 g of LiF reagent and dissolve it in 10 mL of 6 M HCl solution. Stir it at room temperature for 30 min, place it in a constant temperature water bath at 35°C, add 1 g of Ti2AlC MAX powder while stirring, and react for 36 h. The product is centrifuged and washed with ethanol and deionized water in sequence until the pH value of the solution obtained by centrifugation reaches 6. Then, the solid product is vacuum dried at 50°C overnight to obtain Ti2CT x MXene powder; S1-2, Ti2CT x MXene powder was added to dimethyl sulfoxide solvent to prepare a mixture with a concentration of 5 mg / mL. After stirring at room temperature for 24 h, it was centrifuged and washed to obtain a single layer of Ti2CT x MXene suspension; after ultrasonic treatment for 30 min under N2 protection, move it into the reactor, add 150 μL of 25% ammonia water under stirring at room temperature, stir evenly and then hydrothermally react at 120 °C for 6 h. After the reaction, cool to room temperature, filter the product with a 0.22 μm filter membrane and rotary evaporate to obtain MXene quantum dots: MQDs.

4. The method for preparing nanoparticles having both electrocatalytic performance and photoluminescent properties according to claim 1, characterized in that: Step S2 is specifically as follows: S2-1, using a mixed solution of potassium chloride and HCl as a solvent, respectively preparing a MnSO4 solution and a K3[Co(CN)6] solution, and mixing the MnSO4 solution and the K3[Co(CN)6] solution to obtain a mixed solution of MnSO4 and K3[Co(CN)6]; S2-2. The MQDs prepared in step S1 are prepared into an aqueous solution, and then polyvinyl pyrrolidone is added and stirred to prepare a MQD mixed solution containing polyvinyl pyrrolidone, to which a mixed solution of MnSO4 and K3[Co(CN)6] is added dropwise. After the addition is completed, the product is centrifugally washed with deionized water and ethanol in turn, and vacuum dried to obtain PBA@MQDs nanoparticles, i.e., the nanoparticles having both electrocatalytic properties and photoluminescent properties.

5. The method for preparing nanoparticles having both electrocatalytic performance and photoluminescent properties according to claim 4, characterized in that: Step S2 is specifically as follows: S2-1, using a mixed solution of 0.1 M potassium chloride and 0.1 M HCl as a solvent, prepare 50 mL of a 0.005 M MnSO4 solution and 50 mL of a 0.01 M K3[Co(CN)6] solution, respectively, and mix equal volumes of the MnSO4 solution and the K3[Co(CN)6] solution to obtain a mixed solution of MnSO4 and K3[Co(CN)6]; S2-2. Take the MQDs prepared in step S1 to prepare an aqueous solution of MQDs with a concentration of 5 mg / mL. Take 10 mL of the aqueous solution of MQDs, add 0.01 M polyvinyl pyrrolidone thereto, stir for 5-10 min, and prepare a MQDs mixed solution containing polyvinyl pyrrolidone. Add 25 mL of a mixed solution of MnSO4 and K3[Co(CN)6] dropwise thereto, and control the dropwise acceleration rate to 300 μL / min. After the dropwise addition is completed, the product is centrifugally washed with deionized water and ethanol in turn, and dried overnight in vacuum at 60°C to obtain PBA@MQDs nanoparticles, i.e., the nanoparticles having both electrocatalytic properties and photoluminescent properties.

6. A nanoparticle having both electrocatalytic properties and photoluminescent properties, characterized in that: It is prepared by the method according to any one of claims 1 to 5.

7. An application of the nanoparticles having both electrocatalytic properties and photoluminescent properties as claimed in claim 6 in miRNA detection, the application method being: The electrode is modified by using the nanoparticles having both electrocatalytic properties and photoluminescent properties as described in claim 6, and then the capture probe probe designed for the target miRNA is modified to obtain a probe-NH2-PBA@MQDs modified electrode, and then the probe-NH2-PBA@MQDs modified electrode is used to detect the target miRNA by an electrochemical method.

8. Use of the nanoparticles having both electrocatalytic properties and photoluminescent properties in miRNA detection according to claim 7, characterized in that: The application method is: 1) Preparation of NH2-PBA@MQDs modified electrode: 1-1) adding polyetherimide and the nanoparticles having both electrocatalytic properties and photoluminescent properties as claimed in claim 6 into deionized water, stirring and reacting at room temperature, filtering after the reaction is completed, and vacuum drying to obtain NH2-PBA@MQDs; 1-2) Add NH2-PBA@MQDs to deionized water and disperse by ultrasonic to obtain NH2-PBA@MQDs solution; 1-3) The substrate electrode is pretreated, and then the absorbed NH2-PBA@MQDs solution is dropped onto the surface of the substrate electrode, and after drying, the NH2-PBA@MQDs modified electrode is obtained; 2) Preparation of probe-NH2-PBA@MQDs modified electrode 2-1) Design the corresponding capture probe according to the target miRNA, prepare a probe solution, and then add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide for activation; 2-2) Adding a fixing solution to the probe solution obtained in step 2-1) to obtain a probe modification solution, immersing the NH2-PBA@MQDs modified electrode in the probe modification solution for reaction, cleaning the electrode surface after the reaction, and then dropping a melanin-enriching hormone MCH blocking polypeptide solution on the electrode surface for co-incubation to obtain a probe-NH2-PBA@MQDs modified electrode; 3) Target miRNA detection: The sample solution to be tested was co-incubated with the probe-NH2-PBA@MQDs modified electrode to obtain a detection electrode, which was used as a working electrode. An electrochemical workstation was used for electrochemical analysis, and the concentration of the target miRNA in the sample solution was obtained based on the obtained current signal and a pre-constructed standard curve characterizing the relationship between the current signal and the concentration of the target miRNA.

9. Use of the nanoparticles having both electrocatalytic properties and photoluminescent properties in miRNA detection according to claim 8, characterized in that: The application method is: 1) Preparation of NH2-PBA@MQDs modified electrode: 1-1) adding polyetherimide and the nanoparticles having both electrocatalytic properties and photoluminescent properties as claimed in claim 6 into deionized water to prepare a mixed solution with a polyetherimide concentration of 5 mg / mL and a nanoparticle concentration of 1 mg / mL, stirring at room temperature for 4 h, filtering, and vacuum drying at 50°C for 12 h to obtain NH2-PBA@MQDs; 1-2) Add NH2-PBA@MQDs to deionized water and disperse by ultrasonic to prepare a NH2-PBA@MQDs solution with a concentration of 1 mg / mL; 1-3) The Au electrode was used as the base electrode, polished until the surface was smooth, rinsed with deionized water, and dried in N2 atmosphere. Then 10 μL of NH2-PBA@MQDs solution was dripped on the surface of the Au electrode. After natural drying, the solution was repeatedly dripped once to obtain the NH2-PBA@MQDs modified electrode. 2) Preparation of probe-NH2-PBA@MQDs modified electrode 2-1) Design the corresponding capture probe according to the target miRNA, prepare a probe solution with a concentration of 5 μM using MES buffer, then add 0.3 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.6 M N-hydroxysuccinimide, and activate at 4°C for 2 h; 2-2) Add 5 μL of fixing solution to 5 μL of 5 μM probe solution to obtain probe modification solution, immerse the NH2-PBA@MQDs modified electrode in the probe modification solution and react at 4°C for 12 h, then use 10 mM Tris-HCl solution with pH=7.4 to clean the electrode surface, drop 5 μL of 10 mM melanin-enriching hormone MCH blocking polypeptide solution on the electrode surface, incubate at 4°C for 1 h, and obtain probe-NH2-PBA@MQDs modified electrode; The components of the fixative solution were 10 mM Tris-HCl, 1 mM EDTA, and 0.1 M NaCl, pH = 7.4; 3) Target miRNA detection: The sample solution to be tested was incubated with the probe-NH2-PBA@MQDs modified electrode at 37°C to obtain a detection electrode. The detection electrode was used as a working electrode. Electrochemical analysis was performed using an electrochemical workstation. The concentration of the target miRNA in the sample solution was obtained based on the obtained current signal and a pre-constructed standard curve characterizing the relationship between the current signal and the concentration of the target miRNA.

10. Use of the nanoparticles having both electrocatalytic properties and photoluminescent properties in miRNA detection according to any one of claims 7 to 9, characterized in that: The target miRNA was miRNA-141, whose sequence was: 5′-UAACACUGUCUGGUAAAGAUGG-3′; The sequence of the capture probe probe for the target miRNA is: 5′-COOH-CCATCTTTACCAGACAGTGTTA-3′.

11. An application of the nanoparticles having both electrocatalytic properties and photoluminescent properties as claimed in claim 6 in cell imaging, the application method being: The capture probe probe designed for the target miRNA is modified on the nanoparticles having both electrocatalytic properties and photoluminescent properties as described in claim 6 to obtain nucleic acid aptamer-modified nanoparticles probe-NH2-PBA@MQDs, and then the nucleic acid aptamer-modified nanoparticles are used as dyes for fluorescent imaging of the target miRNA in cells.

12. The use of the nanoparticles having both electrocatalytic properties and photoluminescent properties in cell imaging according to claim 11, characterized in that: The aptamer-modified nanoparticle probe-NH2-PBA@MQDs is prepared by the following method: The corresponding capture probe was designed according to the target miRNA, and a probe solution with a concentration of 5 μM was prepared using MES buffer, followed by the addition of 0.3 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.6 M N-hydroxysuccinimide, and activated at 4°C for 2 h; Add 5 μL of the fixing solution to 5 μL of the 5 μM probe solution to obtain a probe modification solution, add 1 mg / mL of the nanoparticles having both electrocatalytic properties and photoluminescent properties according to claim 6 to the obtained probe modification solution, react for 12 h, then wash the obtained nanoparticles with 10 mM Tris-HCl solution, and then incubate the nanoparticles with 5 μL of 10 mM MCH solution for 1 h to obtain nucleic acid aptamer-modified nanoparticles probe-NH2-PBA@MQDs; The components of the fixative solution are 10 mM Tris-HCl, 1 mM EDTA and 0.1 M NaCl, pH=7.

4.

13. Use of the nanoparticles having both electrocatalytic properties and photoluminescent properties in cell imaging according to claim 11 or 12, characterized in that: The target miRNA was miRNA-141, whose sequence was: 5′-UAACACUGUCUGGUAAAGAUGG-3′; The sequence of the capture probe probe for the target miRNA is: 5′-COOH-CCATCTTTACCAGACAGTGTTA-3′.

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